Free-living amoebae prey upon diverse species of microbes in nature, including some that infect humans. The antagonistic dynamics of this process-microbial resistance and amoeba counter-resistance-has led to speculation that amoebae are important reservoirs and even "training grounds" for human pathogens. The broad objective of this research is to use natural variation as a tool for understanding the evolution of innate immune recognition by amoebae and for characterizing the genes and pathways involved. The specific goals of this research proposal are: (1) to take candidate recognition genes identified in a laboratory strain and look for evidence that they are variable in natural populations of amoebae and evolving under positive selection, and (2) to identify the phenotypic effects of this variation by introducing a subset of these genes into a standard laboratory strain and characterizing their effects using molecular genetic techniques. This work is expected to aid in the establishment of the social amoeba Dictyostelium discoideum as a model system for the study of phagoctyosis and innate immunity and to lead to the development of molecular tools and techniques that can be applied to the characterization of genetic variation segregating in nature. More generally, understanding natural variation in the ability to prey upon (or resist infection by) different microbial species can provide insight into the mechanisms that are used by human pathogens to escape phagoctyosis or elude detection by the immune system.